Background:The majority of breast cancers (BCs) expressing estrogen receptor (ER) have shown endocrine resistance. Our previous study demonstrated that ferredoxin reductase (FDXR) promoted mitochondrial function and ER+ breast tumorigenesis. But the underlying mechanism is not clear.Methods:Liquid chromatography (LC) tandem mass spectrometry (MS/MS)-based metabolite profiling was utilized to reveal the metabolites regulated by FDXR. RNA microarray was utilized to determine the potential downstream targets of FDXR. Seahorse XF24 analyzer was performed to analyze the FAO-mediated oxygen consumption rate (OCR). Q-PCR and western blotting assays were used to measure expression levels of FDXR and CPT1A. MTS, 2D colony formation and anchorage-independent growth assays were used to evaluate the effects of FDXR or drug treatments on tumor cell growth of primary or endocrine-resistant breast cancer cells.Results:We found that depletion of FDXR inhibited fatty acid oxidation (FAO) by suppressing CPT1A expression. Endocrine treatment increased the expression levels of both FDXR and CPT1A. Further, we showed that depletion of FDXR or FAO inhibitor etomoxir treatment reduced primary and endocrine-resistant breast cancer cell growth. Therapeutically, combining endocrine therapy with FAO inhibitor etomoxir synergistically inhibits primary and endocrine-resistant breast cancer cell growth.Discussion:We reveal that the FDXR-CPT1A-FAO signaling axis is essential for primary and endocrine-resistant breast cancer cell growth, thus providing a potential combinatory therapy against endocrine resistance in ER+ breast cancer.

Extended methods and supplementary figures

Quantification of IHC staining

Purpose: Metastatic cervical cancer has a poor prognosis, and treatment options are limited. Immunotherapy has been used to achieve disease control in patients with cervical cancer; however, the efficacy of immunotherapy retreatment after disease progression is unclear. This study aimed to explore the efficacy and safety of immunotherapy retreatment in metastatic cervical cancer. Patients and methods: We retrospectively reviewed the clinical data of patients with metastatic cervical cancer who underwent immunotherapy retreatment after disease progression following previous immunotherapy from June 2019 to April 2021.Results: Fifteen patients were included in this study. All patients received combination immunotherapy retreatment consisting of camrelizumab, nab-paclitaxel, and apatinib. Four (26.7%) patients achieved partial response while three (20.0%) achieved stable disease. The objective response rate and disease control rate were 26.7% and 46.7%, respectively. The median progression-free survival and overall survival were 3.0 (95% confidence interval: 1.0-5.0) and 8.0 (95% confidence interval: 3.4-12.6) months, respectively. None of the patients discontinued treatment because of intolerable toxicity.Conclusion: Our findings suggest that the triplet combination immunotherapy retreatment could be a therapeutic option for patients with metastatic cervical cancer who failed initial immunotherapy.

Abstract TANK binding kinase 1 (TBK1) is an important kinase involved in the innate immune response. Here we discover that TBK1 is hyperactivated by von Hippel-Lindau (VHL) loss or hypoxia in cancer cells. Tumors from patients with kidney cancer with VHL loss display elevated TBK1 phosphorylation. Loss of TBK1 via genetic ablation, pharmacologic inhibition, or a new cereblon-based proteolysis targeting chimera specifically inhibits VHL-deficient kidney cancer cell growth, while leaving VHL wild-type cells intact. TBK1 depletion also significantly blunts kidney tumorigenesis in an orthotopic xenograft model in vivo. Mechanistically, TBK1 hydroxylation on Proline 48 triggers VHL as well as the phosphatase PPM1B binding that leads to decreased TBK1 phosphorylation. We identify that TBK1 phosphorylates p62/SQSTM1 on Ser366, which is essential for p62 stability and kidney cancer cell proliferation. Our results establish that TBK1, distinct from its role in innate immune signaling, is a synthetic lethal target in cancer with VHL loss. Significance: The mechanisms that lead to TBK1 activation in cancer and whether this activation is connected to its role in innate immunity remain unclear. Here, we discover that TBK1, distinct from its role in innate immunity, is activated by VHL loss or hypoxia in cancer. See related commentary by Bakouny and Barbie, p. 348. This article is highlighted in the In This Issue feature, p. 327

Immune evasion is essential for carcinogenesis and cancer progression. Programmed death-ligand 1 (PD-L1), a critical immune checkpoint molecule, interacts with programmed death receptor-1 (PD-1) on immune cells to suppress anti-tumor immune responses. In the past decade, antibodies targeting PD-1/PD-L1 have tremendously altered cancer treatment paradigms. Post-translational modifications have been reported as key regulators of PD-L1 expression. Among these modifications, ubiquitination and deubiquitination are reversible processes that dynamically control protein degradation and stabilization. Deubiquitinating enzymes (DUBs) are responsible for deubiquitination and have emerged as crucial players in tumor growth, progression, and immune evasion. Recently, studies have highlighted the participation of DUBs in deubiquitinating PD-L1 and modulating its expression. Here, we review the recent developments in deubiquitination modifications of PD-L1 and focus on the underlying mechanisms and effects on anti-tumor immunity.

Clear cell renal cell carcinoma (ccRCC) is characterized by loss of tumor suppressor Von Hippel Lindau (VHL) function, which leads to accumulation of hypoxia inducible factor α (including HIF1α and HIF2α). HIF2α was previously reported to be one of the major oncogenic drivers in ccRCC, however, its therapeutic targets remain challenging. Here we performed a deubiquitinase (DUB) complementary DNA (cDNA) library binding screen and discovered that ubiquitin-specific peptidase 37 (USP37) is a DUB that binds HIF2α and promotes HIF2α deubiquitination. As a result, USP37 promotes HIF2α protein stability in an enzymatically dependent manner, and depletion of USP37 leads to HIF2α down-regulation in ccRCC. Functionally, USP37 depletion causes decreased cell proliferation measured by MTS, two-dimensional (2D) colony formation as well as three-dimensional (3D) anchorage- independent growth. USP37 is also essential for maintaining kidney tumorigenesis in an orthotopic xenograft model and its depletion leads to both decreased primary kidney tumorigenesis and spontaneous lung metastasis. Our results suggest that USP37 is a potential therapeutic target in ccRCC.

TANK binding kinase 1 (TBK1) is an important kinase involved in the innate immune response. Here we discover that TBK1 is hyperactivated by von Hippel-Lindau (VHL) loss or hypoxia in cancer cells. Tumors from patients with kidney cancer with VHL loss display elevated TBK1 phosphorylation. Loss of TBK1 via genetic ablation, pharmacologic inhibition, or a new cereblon-based proteolysis targeting chimera specifically inhibits VHL-deficient kidney cancer cell growth, while leaving VHL wild-type cells intact. TBK1 depletion also significantly blunts kidney tumorigenesis in an orthotopic xenograft model in vivo. Mechanistically, TBK1 hydroxylation on Proline 48 triggers VHL as well as the phosphatase PPM1B binding that leads to decreased TBK1 phosphorylation. We identify that TBK1 phosphorylates p62/SQSTM1 on Ser366, which is essential for p62 stability and kidney cancer cell proliferation. Our results establish that TBK1, distinct from its role in innate immune signaling, is a synthetic lethal target in cancer with VHL loss. SIGNIFICANCE: The mechanisms that lead to TBK1 activation in cancer and whether this activation is connected to its role in innate immunity remain unclear. Here, we discover that TBK1, distinct from its role in innate immunity, is activated by VHL loss or hypoxia in cancer.See related commentary by Bakouny and Barbie, p. 348.This article is highlighted in the In This Issue feature, p. 327.

Inactivation of the von Hippel-Lindau (VHL) E3 ubiquitin ligase protein is a hallmark of clear cell renal cell carcinoma (ccRCC). Identifying how pathways affected by VHL loss contribute to ccRCC remains challenging. We used a genome-wide in vitro expression strategy to identify proteins that bind VHL when hydroxylated. Zinc fingers and homeoboxes 2 (ZHX2) was found as a VHL target, and its hydroxylation allowed VHL to regulate its protein stability. Tumor cells from ccRCC patients with VHL loss-of-function mutations usually had increased abundance and nuclear localization of ZHX2. Functionally, depletion of ZHX2 inhibited VHL-deficient ccRCC cell growth in vitro and in vivo. Mechanistically, integrated chromatin immunoprecipitation sequencing and microarray analysis showed that ZHX2 promoted nuclear factor κB activation. These studies reveal ZHX2 as a potential therapeutic target for ccRCC.

Objective To develop a probe for photoacoustic imaging and fluorescence imaging targeting integrin αvβ6 . Methods The probe was separated by RP‐HPLC .Molecular weight and the maximum absorption wavelength of the probe were detected by mass spectrum instrument and optical spectrum instrument . Various concentrations of the probe were detected by photoacoustic imaging and fluorescence imaging . The stability of the probe was evaluated when exposed under laser . Targeting of the probe on integrinαvβ6 was evaluated in cell uptake assay with integrinαvβ6 positive and negative cells . The minimum number of cells that could be detected by photoacoustic imaging and fluorescence imaging was also evaluated . Results The probe ICG‐peptide was separated from reaction mixture by RP‐HPLC .The probe had a retention time of 21 .4 minutes and m/z of 4 727 . The labeling ratio of the probe was 1∶1 . The maximum absorption wavelength of the probe was 790 nm . The photoacoustic signal was linearly dependent on the concentration of the probe . The fluorescence signal was linearly dependent on the concentration of the probe when the concentration was smaller than 1 .5 × 10 -5 mol/L . The lowest concentration of the probe that could be detected above the background by photoacoustic imaging and fluorescence imaging was 0 .09 × 10-5 mol/L and 0 .05 × 10-5 mol/L ,respectively . No obvious decrease of the photoacoustic signal was observed after the probe was scanned 20 times ( each time lasted for 1 min) by laser . There existed differences ( P <0 .001) in cell uptake of the probe with various concentrations and reaction time between A431 cells (αvβ6 positive) and 293T cells (αvβ6 negative) . Cell uptake was inhibited by the addition of 5μmol/L unlabeled peptide in A431 cells ( P = 0 .001 ) . The lowest number of the labeled A431 cells detected by photoacoustic imaging and fluorescence imaging was 0 .4 × 106 and 0 .05 × 106 ,respectively . Conclusions The dual functional photoacoustic and fluorescence probe targeting integrin αvβ6 was successfully developed . The targeting and sensitivity of the probe makes it potentially useful in early detection of αvβ6 positive tumors .

The pluronic block copolymers are able to enhance the ultrasound-induced gene delivery in vitro. In the present study, the effects of pluronics on the efficiency of gene transfer into skeletal muscle in vivo under sonoporation were investigated. Plasmid DNA encoding green fluorescent protein (GFP) in combination with three different pluronics, F127, L61, and P85, was injected into the tibialis anterior (TA) muscle of mice with and without adjunct ultrasound (1 MHz, 3 W/cm(2) 1 min, 20% duty cycle). Mice were killed 1 week after injection. The TA muscles were removed and snap frozen immediately in isopentane cooled by liquid nitrogen and sections of 7 μm thick were cut. Transfection efficiency was assessed by counting the number of GFP-positive fibers under fluorescence microscopy, and tissue damage by hematoxylin and eosin staining. The results suggested that all three pluronics significantly enhanced transgene expression in skeletal muscle (P < 0.01), especially the P85 showed significantly higher efficiency than the other two pluronics (P < 0.05). Ultrasound synergistically enhanced the gene delivery efficiency with P85 (P < 0.01), but was unable to do so with F127 and L61 groups. In short, P85 displays significantly synergistic effect with ultrasound for enhancing plasmid DNA transduction in skeletal muscle of mice in vivo.

Pluronic block copolymers, a kind of non-ionic surfactant, also known as poloxamers, and ultrasound-targeted microbubble destruction have been respectively investigated as vectors for gene delivery in vitro and in vivo. However, they are limited for clinical application due to the relatively low transfer efficiency of each individual vector. In the present study, we explored if the combination of P85, a pluronic block copolymer, Optison, a microbubble contrast agent and ultrasound enhances the transfection of plasmid DNA in vivo using mouse skeletal muscle models. Plasmid encoding green fluorescent protein (GFP) was respectively conjugated with 0.05%P85, 10%Optison, or 0.05%P85 plus 10%Optison, and injected into mouse tibialis anterior (TA) muscles with or without ultrasound irradiation (1 MHz, 1 W/cm(2), 2 min and 20% duty cycle). Mice were sacrificed 1 week after injection. The TA muscles were collected and cryo-sectioned into a series of 7 μm slices. To assess the efficiency of plasmid DNA transfection, tissue sections were counterstained with DAPI and scored by counting the number of GFP-positive fibers. Meanwhile the area of damaged muscles was measured based on the tissues stained with hematoxylin and eosin. Both P85 and Optison significantly enhanced the delivery of plasmid DNA in mouse TA skeletal muscles (P<0.01 and P<0.05 respectively, compared to saline control). In combination with Ultrasound irradiation, P85 (P<0.01, compared to P85 alone) but not Optison (P>0.05, compared to Optison alone) exerted a more pronounced effect on the transfection efficiency. Furthermore P85-induced gene delivery was higher than that by Optison regardless of the presence of ultrasound (P<0.01). The highest transfection efficiency was observed when P85, Optison and ultrasound irradiation were administrated together (P<0.01, compared to any other treatment in this study). The area of damaged muscles was enlarged by ultrasound irradiation in the presence of Optison microbubbles (P<0.01, compared to those groups without ultrasound irradiation). These results suggest that P85, microbubbles and ultrasound irradiation synergistically enhance plasmid DNA delivery in mouse skeletal muscles in vivo.

Objective To explore the effects of P85,microbubbles and ultrasound on plasmid DNA skeletal muscle gene transduction of mice in vivo. Methods Plasmid encoding green fluorescent protein (GFP) ,which conjugated with 0.05% P85 and/or microbubbles, 10% Optison,was injected into the tibialis anterior(TA) muscle of mice with or without ultrasound irradiation (1 MHz, 1 W/cm2 2 min,20% duty cycle). Mice were killed 1 week after injection. The TA muscles were removed and snap-frozen immediately in isopentane cooled by liquid nitrogen and sections 7 μm thick were cut at intervals. One set of sections mounted with DAPI were used to assess the transfection efficiency by counting the number of GFP-positive fibers under fluorescence microscopy,and the other set of sections were stained with haematoxylin and eosin to assess the tissue damage area. Results The P85 and Optison significantly enhanced the plasmid DNA skeletal muscle gene delivery in vivo separately (P<0.01, P<0.05).Ultrasound exposure could significantly enhance the efficiency of P85 induced gene delivery(P<0.01) but not of Option(P>0.05).The gene delivery efficiency induced by P85 was higher than that by Optison no matter with or without ultrasound irradiation(P<0.01). When the P85 conjugated with Optison, they could further significantly enhance gene delivery efficiency with ultrasound exposure (P<0.01). Meanwhile, ultrasound exposure could increase the muscle damage areas in the groups with microbubbles (P<0.01). Conclusions The P85,microbubbles and ultrasound exposure display synergistic effect to enhance plasmid DNA transduction in skeletal muscle of mice in vivo.

Objective To observe the characteristics of small hepatic lesions(≤3 cm)with contrast-enhanced ultrasound(CEUS)and evaluate the role of CEUS on the diagnosis of small liver neoplasms.Methods The appearances on CEUS of 136 patients with 156 lesions were studied retrospectively,including 74 malignant cases and 82 benign ones.The time-intensity curve(TIC)of 142 lesions was obtained with computer-assisted software.The characteristics and the parameters of TIC were analyzed correlated with pathological findings.Results Enhancement was found on 146 cases except necrosis nodus and postoperative scars.All(49 cases)the hepatocellular carcinomas(HCC)enhanced in arterial phase,71.43%(35/49)of them enhanced globally.In parenchymal phase,85.71%(42/49)presented as hole'.Typical thick-ringed enhancement was found on 52.00%(13/25)of the metastases in arterial phase and 88.00%(22/25)of them showed as hole' in parenchymal phase.Of benign lesions enhancement can be global(48.61%),patchy(18.06%)and ringed(33.33%)in arterial phase.In parenchymal phase,the proportion of black hole sign was 1.39%,with 30.56% lower than and 68.06% similar to the liver parenchyma.The appearances of inflammatory pseudotumor and angiomyolipomas on CEUS were rapid wash-in and wash-out,similar to malignant lesions.The acoustic quantified parameters of TIC including the time to arrival,time of enhancement and peak time in benign lesions were significantly later than those of the malignant lesions.The difference of the peak intensities between the two groups was not statistically significant.There was no significant difference between primary hepatic carcinomas and metastases.The above mentioned parameters of inflammatory pseudotumor and angiomyolipomas were earlier than those of other benign lesions.Conclusion CEUS has an important clinical value in qualitative diagnosis of liver lesions.The appearances of some benign lesions are atypical,further diagnosis depends on other imageology technique or needle biopsy.

目的探讨超声和共聚物在质粒DNA对小鼠骨骼肌基因转染中的影响。方法应用三种水溶性不同的共聚物F127,L61和P85,与DNA混合后直接小鼠胫前肌注射,并超声辐照。超声频率为1MHz,脉冲重复频率100Hz,占空系数为20%,用3W/cm 2 声强辐照1min。实验1周后取出胫前肌并快速冰冻切片,荧光显微镜计数表达GFP的转染肌纤维数,苏木精和伊红染色评价肌肉损伤情况。结果三种共聚物均可显著提高质粒DNA的基因转染（P<0.01）,P85的基因转染效率高于其他两种共聚物（P<0.05）。超声虽对单纯质粒、F127和L61组的基因转染无显著促进作用,但与P85有协同作用,可显著提高P85组的基因转染效率（P<0.01）。在给定超声剂量和共聚物浓度下,二者在提高质粒DNA基因转染的同时,并不显著增加对组织的损伤作用。结论共聚物P85不但可显著提高质粒DNA在小鼠骨骼肌的基因转染,且与超声具有协同作用。

目的 探讨超声和共聚物在不同细胞系中对基因转染的协同作用.方法 应用三种水溶性不同的共聚物F127,L61和P85;超声频率为1 MHz,脉冲重复频率100 Hz,占空系数为20%,用1 w/cm2声强照射20 s.选用C2C12,3T3-MDEI,CHO和H2K四种细胞系为研究对象.质粒DNA为可产生绿色荧光蛋白的GFP.应用荧光显微镜和流式细胞仪评价转染率,台盼蓝染色评价细胞的成活率.结果 超声可介导所有四种细胞系的基因转染.在C2C12,3T3-MDEI和CHO细胞系,共聚物可显著促进超声介导的基因转染效率(P＜0.01),对H2K细胞系共聚物不但不能促进超声介导的基因转染、,其中疏水性共聚物P85和L61反而使超声介导的基因转染效率降低(P＜0.05).结论 共聚物对不同细胞系超声介导的基因转染效率产生不同的影响,这种差异可能与细胞膜蛋白缺陷有关。

To examine the role of ultrasound in gene delivery in vitro, three cells lines were exposed to the low-frequency ultrasound of varying intensities and for different durations to evaluate their effect on gene transfection and cell viability of the cells. Microbubble (MB), Optison (10%), was also used to observe the role of the microbubbles in gene transfection. The results demonstrated that as the ultrasound intensity and the exposure time increased, the gene transfer rate increased and the cell viability decreased, but at high energy intensities, the cell viability decreased dramatically, which caused the transfer rate to decrease. The most efficient ultrasound intensity for inducing gene transfer was 1 W/cm(2) with duration being 20 s. At the same energy intensity, higher ultrasound intensity could achieve maximal gene transfer rate earlier. Microbubbles could increase ultrasound-induced cell gene transfer rate by about 2 to 3 times mainly at lower energy intensities. Moreover, microbubbles could raise the maximum gene transfer rate mediated by ultrasound. It is concluded that the low-frequency ultrasound can induce cell gene transfer and the cell gene transfer rate and viability are correlated with not only the ultrasound energy intensity but also the ultrasound intensity, the higher ultrasound intensity achieves its maximal transfer rate more quickly and the ultrasound intensity that can induce optimal gene transfer is 1 W/cm(2) with duration being 20 s, and microbubbles can significantly increase the maximal gene transfer rate in vitro.

To investigate the functions of Pluronics block copolymers in ultrasound induced cell damage and gene delivery,three different Pluronics block copolymers F127,L61 and P85 at different concentrations were studied in three cell lines(C2C12,3T3-MDEI,and CHO-E).The results indicated that the ultrasound could induce cell damage and the viability declined as increasing ultrasound power.At low concentration around CMC,the Pluronics block copolymers could enhance ultrasound mediated cell damage(P0.05)and gene delivery(P0.01).But the Pluronics block copolymers behaved differently at high concentration above 10 to 20 folds of CMC,some of which,like F127 and P85,could protect against ultrasound induced cell damage even when the power of the ultrasound was increased 10-folds,while the others,like L61,showed high toxicity.At the same time,they could not enhance ultrasound induced gene delivery at high concentrations.In short,Pluronics block copolymers could enhance ultrasound mediated gene delivery at low concentrations and some of which could protect against ultrasound mediated cell damage at high concentrations.

In order to assess whether gene transfection could be mediated by ultrasound in association with P85 and find the appropriate parameters of ultrasound irradiation, the effects of ultrasound with or without P85 on gene transfection of HepG2 cells were examined. The HepG2 cells were irradiated by ultrasound at 1 MHz, 0.4–2.0 W/cm2 and 50% duty cycle with plasmid encoding enhanced green fluorescent protein (EGFP) as a report gene. Forty-eight h later, the expression of EGFP was detected under the fluorescence microscopy. Transfection efficacy was quantitatively assessed by flow cytometry, and cell viability was evaluated by trypan blue exclusion. The results showed that the transfection efficacy was increased with the increases in ultrasound output power and the ideal transfection efficacy was achieved in HepG2 cells irradiated by ultrasound at 0.8 W/cm2 for 30 s. The transfection efficacy in ulstrasound+P85 group was three times higher than in single ultrasound group [(17.63±1.07)% vs (5.57±0.56)%, P<0.05]. The cell viability was about 81% and 62% in ultrasound group and ultrasound+P85 group respectively. It was concluded that ultrasound in combination with P85 could mediate the gene transfection of HepG2 cells, ideal transfection efficacy was achieved by ultrasound irradiation at 0.8 W/cm2 for 30 s, and P85 could somewhat increase the damage to cells caused by ultrasound.